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Nucleic Acids Research Advance Access published online on May 19, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp383
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© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited

Methods Online

Quantitative evaluation of DNA methylation by optimization of a differential-high resolution melt analysis protocol

Francesca Malentacchi, Giulia Forni, Serena Vinci and Claudio Orlando*

Clinical Biochemistry Unit, Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, 50139, Florence, Italy

*To whom correspondence should be addressed. Tel: +39 55 4271440; Fax: +39 55 4271413; Email: c.orlando{at}dfc.unifi.it

Received September 18, 2008. Revised April 27, 2009. Accepted April 27, 2009.

DNA methylation is a key regulator of gene transcription. Alterations in DNA methylation patterns are common in most cancers, occur early in carcinogenesis and can be detected in body fluids. Reliable and sensitive quantitative assays are required to improve the diagnostic role of methylation in the management of cancer patients. Here we present an optimized procedure, based on differential-high resolution melting analysis (D-HRMA), for the rapid and accurate quantification of methylated DNA. Two sets of primers are used in a single tube for the simultaneous amplification of the methylated (M) and unmethylated (Um) DNA sequences in D-HRMA. After HRM, differential fluorescence was calculated at the specific melting temperature after automatic subtraction of UM-DNA fluorescence. Quantification was calculated by interpolation on an external standard curve generated by serial dilutions of M-DNA. To optimize the protocol, nine primer sets were accurately selected on the basis of the number of CpG on promoters of hTERT and Bcl2 genes. The use of optimized D-HRMA allowed us to detect up to 0.025% M-DNA. D-HRMA results of DNA from 85 bladder cancers were comparable to those obtained with real time quantitative methylation specific PCR. In addition, D-HRMA appears suitable for rapid and efficient measurements in ‘in vitro’ experiments on methylation patterns after treatment with demethylating drugs.


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